PRACTICE REPORT

MICROBIOLOGY
Author: Dr. Elmar Grabert, HACH LANGE GmbH, Germany

Microbiological tests –
not just for safe drinking water
Summary The analysis of microorganisms is an indispensable ele- Bacteria are simply everywhere
– so naturally they are also pre-
ment of modern hygiene and disinfection measures. The success of sent in water. They can pose a
these measures can only be assessed using microbiological tests. risk to health, and often form
To be able to intervene as early as possible, quick screening results biofilms on surfaces. Damage may
then be caused by bacterial corro-
are especially important, as standard analyses are usually cost in- sion or mechanical defects in
tensive and simply take too long. Here is an overview of the microbi- pipes. In order be able to respond
adequately to these uninvited
ological tests and their uses: guests, they must be identified
with a suitable test system. HACH
Aerobic bacteria or moulds? LANGE tests have already proven
PADDLE testers show in just 24 hours whether surfaces or liquids themselves in many applications –
have been properly disinfected. They are also suitable for applica- they are easy to carry out and give
tions in the process water, paper, dye and paint industries and in reliable results.
cutting fluids.
Faecally contaminated or not?
The answer is supplied by P/A (Presence/Absence) tests. E. coli
bacteria serve here as an indicator of really dangerous faecal organ-
isms, e.g. Cholera pathogens. The P/A test measures the E. coli
LABORATORY ANALYSIS_MICROBIOLOGY

concentration with an accuracy of one organism in 100 ml sample.

E. coli or coliforms?
In just 24 hours, the M COLI BLUE24 membrane filtration test determines safely and reliably
whether drinking water contains these bacteria, and if so, how many. This is especially important
when the number of organisms plays a crucial role, as in the pharmaceutical and cosmetic sectors,
and in surface and bathing waters.
Getting to the source of the problem?
A blocked filter, a poorly performing well, unpleasant odours – in all these cases, BART tests iden-
tify the bacterial culprits quickly and unambiguously.

Complete Report: Microorganism-free drinking and process water?

Fast microbiological tests give the answer
Introduction
Microorganisms, also referred to
as microbes, are the smallest
living organisms that are visible
through a microscope. Bacteria,
unicellular algae and moulds,
yeasts and protozoa are all
microorganisms.
Bacterium in focus
Bacteria form the largest group of
microorganisms. They have an
amazing ability to survive under
almost any conditions – with or
without air, in darkness or in light,
in water, oil or absolutely dry con-
ditions, in or on other living organ-
isms. It is therefore no surprise
that bacteria can be found almost
everywhere.
Depending on their type and
mode of life, bacteria may be
beneficial or they may harm hu-
man health, food, consumables or
technical equipment. Legally bind-
ing regulations therefore exist,
whose purpose is to prevent un-
desirable contact with pathogenic
microorganism.
Prevent harms
Examples of such harmful effects
are the fouling of process water
and cooling water circuits, bacte-
rial corrosion of pipes or coated
cans of preserved food, and the
impairment of the stability of pa-
per, dyes and paint products.
Today an immense number of
widely varied methods exist for
detecting microorganisms. The
most important microbiological
tests are listed by application and
described below.
Classification of test methods
General determination of
aerobic bacteria and
moulds:

Viruses are not microorganisms; Strict hygienic regulations A) PADDLE testers

they cannot reproduce
independently but are dependent
on the metabolic machinery of
their host cells, and are therefore
not regarded as “living”.
All microorganisms play an
important role in maintaining
nature’s metabolic balance,
through their ability to convert
(mineralise) biomass. They do
this by using large, complex
organic molecules and small
inorganic molecules as sources of
nutrients and breaking down the
chemical bonds to create smaller,
simpler structures.
These regulations stipulate which
microorganisms must not be pre- Determination of indicator
sent, or may only be present up to organisms (organisms
a given limit. Hygiene regulations that indicate the possible
also define the individual meas-
presence of pathogens):
ures that must be taken to prevent
microbial contamination.
B1) presence/absence (P/A)
test
Examples are the Drinking Water
Act, Mineral and Table Water Act,
B2) membrane filtration test
Milk Act, European Bathing Water
with M COLI BLUE24
Directive and the HACCP (Hazard
Analysis of Critical Control Point)
system. Determination of bacteria
that can harm technical
Outside the scope of such legal equipment:
regulations, measures are also
needed to prevent the harmful C) BART tester
effects of microbial action.
LABORATORY ANALYSIS_MICROBIOLOGY
General determination
of aerobic bacteria and
moulds:
A) PADDLE testers
PADDLE testers are a simple
way of determining within 24
hours whether large or small
numbers of bacteria, fungi or
moulds are present in a sample or
on a surface.
One of the paddle’s two sides is
red, and indicates the presence of
bacteria, while the other (yellow)
side detects yeasts and moulds.
On the one side is a trypton glu-
cose extract agar (yellow, for the
total organism count) and the
other has a coating of a selective
agar, which depends on the type
of tester.
The paddle is either dipped in the
sample or pressed against the
bacteriologically contaminated
surface. It is then returned to the
vial in which it was supplied and
then incubated for 24 hours at 35-
Fig. 1: Incubator for 24h incuba-
tion, and optical evaluation of a
paddle tester.
37°C (see Fig. 1).
The paddle is then evaluated
optically. Red spots indicate the
presence of colonies of bacteria.
If the red spots are densely
packed, there are correspondingly
large numbers of bacteria in the
sample, and if there are only a
few or no spots, the level of bac-
terial contamination is low. The
measuring and detection range of
the paddle tester is approximately
102 to 107 organisms in 1 ml sam-
ple.
Reliable checking
The paddle tester is eminently
suitable for checking surfaces or
liquids that have been disinfected.
If the findings are positive, this
indicates a less than optimal
disinfection result. If the paddle
remains free of red spots, the
disinfection was carried out
correctly.
The paddle tester can also be
used to good effect in other areas
where bacteria can theoretically
multiply but are basically undesir-
able, such as cooling water cir-
cuits, process water in the paper,
dye and paint industries or in cut-
ting fluids in the metal processing
sector. PADDLE testers are of
limited efficacy, however, for use
with viscous liquids.
Determination of
indicator organisms
(organisms that indicate
the possible presence
of pathogens):
E. coli
Worldwide, the most widely
tested-for bacteria are E. coli (Es-
cherichia coli) and coliforms. Es-
cherichia coli is a rod-shaped
bacterium with a length of 2-4 µm
and a diameter of 1 µm. It occurs
naturally as a harmless inhabitant
of the human intestine. Since E.
coli can also survive for a certain
length of time outside the intes-
tine, however, and can also be
easily detected, it serves as an
indicator of faecal contamination
in water, and especially in drink-
ing water.
A positive identification of these
bacteria in water always indicates
contamination of the sample with
intestinal bacteria. In principle,
there is always a possibility that
these relatively harmless bacteria
are accompanied by really dan-
gerous pathogens such as Sal-
monella, Cholera, or intestinal
viruses, representing a risk to
human health.
For this reason a number of regu-
lations have been enacted, pro-
hibiting the presence of these
organisms in drinking water. For
instance, 100 ml tap water should
not contain detectable levels of E.
coli or coliforms.
Highly sensitive methods
Methods of analysing E. coli must
therefore be highly sensitive. It
must be absolutely certain that a
negative test result really indi-
cates the absence of E. coli in
100 ml original sample.
As an indication of the level of
sensitivity required, detecting a
bacterium in 100 ml water is
equivalent to finding a grain of rye
in a 200 km long goods train full
of wheat. Two different methods
can be used to detect coliforms
and E. coli.
B1) Presence/absence
tests (P/A tests)
As the name suggests, the pres-
ence/absence (P/A) test can be
used to check for the presence or
absence of coliforms in a water
sample. It is simple to use and the
results are very reliable.
LABORATORY ANALYSIS_MICROBIOLOGY
The test reagent – a presence-
absence broth – is introduced into
a 120 ml sample bottle. 100 ml of
the sample are added and the
bottle is incubated for 24 hours
(or not more than 48 hours) at 35-
37°C (see Fig.2).
Fig. 2: P/A test in incubator
If the original red colour of the
solution does not change, no
coliforms and therefore no E. coli
are present in the sample. A
change of colour to yellow indi-
cates the presence of coliforms or
E. coli.
Reliable Detection
This fast test is a reliable detec-
tion method for use in water
analysis. It gives the operator of a
drinking water plant the option of
quickly obtaining an overview of
the hygiene situation in general
after repairs or direct interven-
tions have been carried out in the
drinking water network. The sen-
sitivity of the P/A test is1 organ-
ism per 100 ml sample.
B2) MEMBRANE
FILTRATION method
A far more exact method for de-
termining the type and number of
specific indicator organisms such
as E. coli, total coliforms, faecal
coliforms, total aerobic organisms
or pseudomonads is membrane
filtration. For this reason, it is also
mainly used in routine analysis.
The ready-to-use test contains
everything that is needed, includ-
ing membrane filters, funnels,
petri dishes, absorbent pads, and
the indicator medium M COLI
BLUE24.
The user only needs to provide a
vacuum pump for the membrane
filtration.
The sample is filtered through the
membrane filter (nitrocellulose,
0.45 µm average pore diameter).
All the bacteria in the sample
remain on the surface of the filter.
The filter is then placed in a petri
dish containing an absorbent pad
which has been soaked with the
indicator medium.
A variety of indicator media are
available to detect different bacte-
ria (e.g.: mColiBlue24 for total
coliforms and E. coli; m-Endo for
total coliforms, m-FC for faecal
coliforms, m-Pseudomonas broth
for pseudomonads and m-HPC
for the total organism count). The
absorbent pad is soaked with the
relevant indicator medium imme-
diately before the test is per-
formed.
1 2
Fig. 3: Membrane filtration. The sample is filtered (1); indicator medium m-ColiBlue24 is
poured onto the absorbent pad (2); the membrane filter is placed on the absorbent pad
and incubation is carried out (3); the result is read (4a): tap water without E. coli, (4b): tap
water with E. coli (1 organism/100 ml).
The dish is then incubated for 24
hours at 35-37°C. Thanks to this
preselection only the target bacte-
ria can multiply on the membrane
filter. Their presence is betrayed
by variously coloured spots on the
white filter. Figure 3 shows the
detection of an E. coli organism in
tap water.
The indicator medium M COLI
BLUE24® can therefore be used
to determine, within 24 hours,
either the absence of E. coli or the
exact number of E. coli (blue
spots) and total coliforms (red
spots).
The membrane filtration method
requires relatively little effort and
yields very reliable results. It is
the ideal method for analysing
process water in the pharmaceu-
tical and cosmetic sectors as well
as drinking water and bottled wa-
ter, and for monitoring surfaces
and bathing water, where the
exact organism count is crucial.
3 4a 4b
LABORATORY ANALYSIS_MICROBIOLOGY
Determination of
bacteria that can cause
damage to technical
equipment:
Wherever technical equipment
comes into contact with liquids, as
in pumps, pipelines, tank farms
and storage facilities, but also in
drinking water wells, bacteria
cause undesirable effects such as
corrosion, discolouration, block
of suspended solids, flocking, and
changes of taste and odour.
C) BART tester
A reliable aid to the prompt identi-
fication of these bacteria is the
BART™ tester (see Fig.4).
Fig. 4: Three BART tests
Universal use
The Biological Activity Reaction
Test (BART™) is a water test for
a variety of bacteria whose pres-
ence in water is undesirable. It is
available as a number of test kits.
These determine the activity of
the bacteria in the water by the
time that is needed for an easily
visible reaction in the test system.
The longer the time before the
reaction can be observed, the
lower the level of biological activ-
ity. The evaluation is carried out in
two steps.
First of all the biological
activity is determined by the time
that elapses before a certain reac-
tion is observed, and secondly the
bacterial population that is pre-
sent can be deduced from the
reaction pattern.
The monitoring of drinking water
wells is one application for which
the BART tests are used. During
the use of groundwater wells,
symptoms of ageing appear,
paired with loss of performance.
The diminished efficiency of the
well performance is attributable to
physical effects such as sanding,
fusion and ferric incrustation; of
these, ferric incrustation is often
caused by biological factors.
In order to be able to determine
the right time to regenerate a well,
extensive testing must be carried
out.
Easy to perform
The BART™ test for determining
biological activity in wells and
groundwater is an important aid
here. The BART tests are very
easy to perform.
The tube is filled up to the mark
with the sample. No further mixing
occurs and the tube is incubated
for 7–9 days at room temperature.
The tubes are checked each day
for the appearance of certain re-
actions described in the instruc-
tion leaflet (e.g. colouration, tur-
bidity, slime formation).
The earlier and stronger the reac-
tion, the larger and more aggres-
sive the bacterial population that
causes it. The reaction is not ana-
lysed on a nutrient plate (solid
medium) but in the natural habitat
(sample water).
Seven different test kits
There are seven different test kits.
These tests are specific for iron
related bacteria (RB), sulphate
reducing bacteria (SRB), hetero-
trophic aerobic bacteria (HAB),
slime forming bacteria (SLYM),
nitrifying bacteria (N), denitrifying
bacteria (DN) and fluorescent
Pseudomonads (FLOR). As each
group of bacteria causes different
problems, a combination of tests
is advisable.
Berliner Wasserbetriebe
A BART test was used for a trial
period to monitor a drinking water
well of the Berliner Wasserbe-
triebe.
Samples were taken from a
freshly regenerated well over a
period of weeks and the water, or
rather the bacteria in the water,
were tested using the BART™
test.
Result: faster, more intensive!
As a comparison, parallel controls
were set up with tap water and
sterile tap water. It was observed
that as the period of operation of
the well increased, the reaction to
the BART™ test occurred faster
and became more intensive.
The observed reaction in the tap
water control was very slight and
occurred after some delay. No
reaction was observed in the ster-
ile tap water control.
The results indicated an increas-
ing number of bacteria and in-
creasing aggressiveness of the
bacterial population as the well
operation time increased. In paral-
lel to this, the organism count was
determined with a conventional
dilution series on nutrient plates.
LABORATORY ANALYSIS_MICROBIOLOGY

The evaluation showed that repro-
ducible and comparable results
were obtained for the organism
number on nutrient plates and with
the BART™ test (see Tab.2)
The other studies using BART™
tests and parallel pump tests also
showed that the causes of the drop
in performance of the studied wells
were purely physical and chemical
as well as biological
Summary
Analysis of microorganisms is
now an indispensable part of a
modern and comprehensive
system of hygiene monitoring.
.The results obtained reveal the
success of failure of any hygiene
measures that have been carried
out.
Fast biological tests that can be
easily carried out in the field are
playing an increasingly important
role and quickly provide the user
with the results he needs to re-
spond promptly to events. Costly
and time-consuming routine
analysis often cannot offer this
option.

Sample Plate test BART™ test

“R2A – Agar” “HAB”
[organisms/ml] [organisms/ml]
5
Bacterial solution 5.1 x 10
5 5 x 10

1 1
Groundwater 1 x 10 1 x 10

Tab. 2: Comparison of the determination of the organism count of aerobic bacteria

using the dilution method on nutrient plates and the BART™ test.
Source: Umwelttechnik Dr. Bartetzko GmbH

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